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WO2007016529A2 - Methodes de criblage de composes pour le risque proarhythmique et l'efficacite antiarhythmique - Google Patents

Methodes de criblage de composes pour le risque proarhythmique et l'efficacite antiarhythmique Download PDF

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Publication number
WO2007016529A2
WO2007016529A2 PCT/US2006/029832 US2006029832W WO2007016529A2 WO 2007016529 A2 WO2007016529 A2 WO 2007016529A2 US 2006029832 W US2006029832 W US 2006029832W WO 2007016529 A2 WO2007016529 A2 WO 2007016529A2
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type calcium
calcium channel
test compound
tissue
freshly isolated
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PCT/US2006/029832
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WO2007016529A3 (fr
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Gan-Xin Yan
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Main Line Health Heart Center
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Priority to EP06789048A priority Critical patent/EP1913386A2/fr
Publication of WO2007016529A2 publication Critical patent/WO2007016529A2/fr
Publication of WO2007016529A3 publication Critical patent/WO2007016529A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/326—Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes

Definitions

  • the present invention relates generally to the field of safety pharmacology.
  • the invention features novel methods for screening compounds for their potential to induce a cardiac arrhythmia or to inhibit a cardiac arrhythmia in a subject, as well as methods to generate a risk score for distinguishing among different compounds' capacity to cause torsade de pointes.
  • Ventricular repolarization time is determined by transmembrane action potential duration (APD) of the ventricular myocardium or the QT interval on the body surface electrocardiogram (ECG). Delayed ventricular repolarization that manifests as QT interval prolongation on the ECG is associated with the development of an atypical form of polymorphic ventricular tachycardia termed torsade de pointes (TdP) that can result in recurrent fainting and sudden death in humans. An increasing number of medications are found to prolong ventricular repolarization, leading to QT prolongation. Some of these medicines are developed purposefully to prolong cardiac APD for the treatment of cardiac arrhythmias such as atrial fibrillation and monomorphic ventricular tachycardia.
  • APD transmembrane action potential duration
  • ECG body surface electrocardiogram
  • antiarrhythmic drugs include sotalol, dofetilide and amiodarone.
  • Many such drugs are administered in suboptimal doses to avoid TdP.
  • suboptimal dosing of these drugs in humans in order to avoid TdP greatly attenuates their efficacy in the inhibition of cardiac arrhythmias.
  • non-cardiac agents such as cisapride, terfenadine, erythromycin and sparfloxacin, have been removed from the market or relabeled for restricted use because of their proarrhythmic potential.
  • Recent regulatory guidelines recommend preclinical assessment of potential new drugs for QT prolongation and the resultant risk of TdP in humans.
  • This screening method suffers from several major drawbacks.
  • the drawbacks include the fact that this method is not well suited for high throughput screening, and that TdP risk is not proportional to the potency of a compound to inhibit hERG current.
  • this method has a fairly high potential to produce false negatives and positives. A false negative may result if the hERG channel is not the K + channel target of the compound. False positives may result where the blockage of the hERG channel does not directly correlate with a prolonged QT interval, hi addition to the /kr current, there are other ventricular membrane currents which may be affected by a drug.
  • the ventricular action potential duration which determines the QT interval, is the consequence of a dynamic balance of multiple membrane currents.
  • testing only the effect of a drug on the / kr current may overlook the drug's effect on other membrane currents such as those produced by Calcium or Sodium ion channels, thereby blurring the picture of the drug's effect on the QT interval.
  • the typical examples are verapamil and amiodarone. Verapamil is a potent hERG current inhibitor, yet is not associated with significant QT prolongation and is free of TdP risk in humans (Yang T et al., J. Cardiovasc. Pharmacol. 2001; 38: 737-744).
  • amiodarone inhibits hERG current at fairly low concentrations and significantly prolongs the QT interval, although it rarely causes TdP in humans (Mattioni TA et al. Ann. Intern. Med. 1989/111 : 574-580).
  • a second screening method involves the direct measurement of action potential duration in isolated ventricular Purkinje fibers or ventricular myocardium.
  • the rationale behind this method is the expectation that a compound would be likely to cause TdP if it can be shown to increase action potential duration. (Champeroux, P., et al. Br. J. Pharmacol. 144:376-85 (2005)).
  • This screening method is not a very sensitive assay, and thus is generally done in conjunction with another screen such as hERG inhibition.
  • This screening method also suffers from an additional drawback in that it has a high potential to cause false positive or negative results.
  • false positives include drugs such as amiodarone, which may cause a prolonged action potential duration or QT interval (van Opstal JM et al, Circulation 2001; 104:2722-2727), yet not be potent inducers of TdP.
  • false negatives include the drugs bepridil and terfenadine, which are known to cause TdP in humans, but have not been shown to produce a significant increase in the action potential duration of the Purkinje fiber (Champeroux et al., Br. J Pharmacol. 2005;144:376-385).
  • a third screening method involves the measurement of the QT interval or the duration of monphasic action potential in the isolated, Langendoff-perfused heart. (Hondeghem, L.M., et al, Circulation, 2001 ;103 -.2004-13).
  • This method suffers from a significant drawback as well.
  • the drawback is that the electrical stability of the perfused heart preparations is of relatively short duration, on the magnitude of less than two hours. The instability of the preparations may thus result in false negative or false positive results.
  • the QT interval (or ventricular APD) seems to be positively proportional to body mass among various species under physiological conditions, ranging from tens of milliseconds in mice to hundreds of milliseconds in large animals as shown in Figure 1.
  • APD prolongation in small animals to an extent may lead to occurrence of early afterdepolarization (EAD) capable of initiating TdP, but in larger species, the same or longer APDs can be physiological.
  • the physiological QT interval in the cow is approximately 400 ms, but the same length of QT would be likely associated with a high risk of TdP in the rabbit.
  • EAD early afterdepolarization
  • TdP is triggered by ventricular action potential EAD at repolarization phase 2 or phase 3.
  • EAD as the trigger plays a central role in the development of TdP, and its occurrence in ventricular action potentials could serve as a marker to determine if a prolonged QT interval is physiological or pathophysiological.
  • the L-type calcium channel current (le a ) is the primary charge carrier for phase 2 EADs under delayed ventricular repolarization (Clancy& Rudy., Nature 1999; 400: 566-569; Viswanathan & Rudy., Cardiovasc Res. 1999; 42: 530-542)
  • its availability after the initial activation for the development of EAD during repolarization phase 2 is critical for the development of TdP.
  • the present invention features methods to screen compounds for their potential to induce or inhibit a cardiac arrhythmia.
  • the methods comprise determining the ratio of the time constant ( ⁇ ) of Ic a j L recovery in tissue expressing the L-type calcium channel or any subunit or combination thereof of the L-type calcium channel treated with a test compound to the ventricular repolarization time of cardiac tissue treated with a test compound.
  • the ratio is determined by measuring the ventricular repolarization time of cardiac tissue treated with a test compound, measuring the recovery of the L-type calcium channel current (Ica, L ) in tissue expressing the L-type calcium channel or any subunit of the L-type calcium channel, calculating the time constant ( ⁇ ) of Ic a , L recovery in the tissue expressing the L-type calcium channel or any subunit of the L-type calcium channel, and calculating the ratio of the time constant ( ⁇ ) of Ic a , L recovery to the ventricular repolarization time.
  • the ventricular repolarization time is measured by a glass microelectrode or monophasic action potential electrode or an electrocradiogram or is measured by an electrogram using unipolar or bipolar electrodes
  • the L-type calcium channel recovery is measured by a voltage clamp such as a whole-cell voltage clamp, which can employ a double pulse protocol.
  • the L-type calcium channel recovery can be measured on any cardiac tissue, or any tissue expressing the L-type calcium channel or any subunit or combination of subunits of the L-type calcium channel, such as stable cell lines.
  • test compound is assessed for its effect on the ventricular repolarization time and recovery of the L-type calcium channel current at multiple doses, which span a range from the compound's free therapeutic plasma C max to a concentration equal to or greater than 500-fold over the C max .
  • the methods of the present invention feature determining a TdP risk score by comparing the ratio of the time constant ( ⁇ ) of Ic a , L recovery to the ventricular repolarization time for the test compound to the ratio of the time constant ( ⁇ ) of Ic a,L recovery to the ventricular repolarization time of a standard having an established risk.
  • FIG. 1 Proportion of QT Interval to Body Mass.
  • (a) Original recordings of transmembrane action potentials from endocardium (Endo) and epicardium (Epi) and ECG from the mouse, guinea pig, and rabbit left ventricular wedge preparations
  • (b) Original recordings of transmembrane action potentials from subendocardium (Subendo ) and Epi and ECG from the canine and cow left ventricular wedge preparations.
  • T p-e represents the interval from the peak to the end of T wave.
  • (c) Graphical depiction of the relationship between the action potential duration and left ventricular wall thickness
  • (d) Graphical depiction of the relationship between transmural dispersion of repolarization and left ventricular wall thickness.
  • APD 90 action potential duration at 90 % repolarization
  • EAD early afterdepolarization
  • FIG. 4 Comparison of Dose-dependent Effect on QT Intervals.
  • the dose-dependent effect on the QT interval among quinidine, sotalol, bepridil, haloperidol, moxifloxacin, flecainide, citalopram, loratadine, fluxetine and verapamil in rabbit heart preparations was compared.
  • Isolated rabbit left ventricular wedge preparations were treated with the compounds in a concentration equivalent to the free therapeutic plasma Cmax up to concentrations 100-fold over the Cmax.
  • a transmural ECG was concurrently recorded.
  • TdP Score Using on the criteria in Table 1, below, the TdP scores of test compounds were calculated, and the calculated scores at each dose are shown. The relative TdP are compared among quinidine, sotalol, bepridil, haloperidol, moxifloxacin, flecainide, citalopram, loratadine, fluxetine and verapamil.
  • the maximal TdP score in the testing concentration range is ranked from high to low: quinidine (11.75 ⁇ 0.22), sotalol (lO.OO ⁇ O.OO), bepridil (7.50 ⁇ 0.26), haloperidol (4.20 ⁇ 0.58), moxifloxacin (4.00 ⁇ 0.00), flecainide (1.25 ⁇ 0.22), citalopram (1.25 ⁇ 0.22), fluxetine (O.OO ⁇ O.OO), loratadine (-0.25 ⁇ 0.22) and verapamil (- 2.50 ⁇ 0.26).
  • Relative risk is indicated by background shading: white/no shading corresponds to a negative risk of inducing TdP, light gray corresponds to a low risk, halftone gray corresponds to a moderate risk, and dark gray corresponds to a high risk.
  • proarrhythmic refers to any tendency to induce any variation in the normal physiology or rhythm of a heartbeat in a subject, including without limitation tachycardia, bradycardia, and fibrillation.
  • antiarrhythmic refers to any tendency to correct, restore, or otherwise remedy any variation in the normal physiology or rhythm of a heartbeat in a subject.
  • Repolarization refers to the reestablishment of polarity, especially the return of cell membrane potential to resting potential after depolarization, in excitable tissue such as cardiac tissue.
  • excitable tissue refers to any tissue that can generate an electrical impulse or can be activated by an electrical stimulus. Such tissue include without limitation skeletal muscle, smooth muscle, cardiac tissue, and nervous tissue.
  • Action potential duration refers to the time between the start of depolarization and the end of repolarization of myocytes in the ventricles and ventricle tissue of the heart.
  • action potential duration can be measured using microelectrodes, and the longest action potential duration of ventricles manifests as the QT interval on the electrocardiogram.
  • Long QT or “LQT” refers to any prolongation of the normal time between the start of depolarization and the end of repolarization of the ventricles and ventricle tissue of the heart, which is manifest on the QT interval of an electrocardiogram.
  • Recovery refers to the availability of L-type Ca 2+ channel for reactivation after inactivation.
  • tissue refers to any cell or group of cells.
  • Cardiac tissue refers specifically to any cell or group of cells isolated by any means from the heart of an animal.
  • Stable cell or “stable cell line” refers to any cell in which any subunit of the L- type calcium channel or combinations thereof, including the whole L-type calcium channel, can be expressed so that the kinetics of Ic a , L recovery can be examined.
  • Drug-induced cardiac proarrhythmia and antiarrhythmia are two sides of the same coin.
  • Efficacy of some antiarrhythmic drugs e.g., sotalol, dofetilide and amiodarone, to inhibit cardiac arrhythmias, including atrial fibrillation and monomorphic ventricular tachycardia, is expected to reflect their effect to prolong cardiac action potential duration, i.e., cardiac repolarization time.
  • cardiac action potential duration i.e., cardiac repolarization time
  • an excessively prolonged ventricular repolarization time which manifests as QT interval prolongation on the body surface ECG, is associated with atypical form of polymorphic ventricular tachycardia termedT, resulting in recurrent syncope and sudden death in humans.
  • APD and the inhibition of related ionic currents such as hERG current have been considered as a surrogate of TdP.
  • Clinical data indicate that the risk of TdP is not proportional to QT prolongation.
  • a QT interval can be physiological in one species but pathophysiogical in another.
  • the physiological QT interval in the cow is approximately 400 ms, but the same length of QT would be likely associated with a high risk of TdP in the rabbit. Therefore, it stands to reason that there is a mechanism for stabilizing cell membrane potentials in the large species, such that a normal heart rhythm can be maintained under a relatively longer QT interval.
  • the present invention is derived from the data that indicate an intrinsic balance between Ic a ,L recovery and ventricular myocyte repolarization under physiological conditions in various species.
  • the impairment of this intrinsic balance by drugs either by prolongation of repolarization time or by acceleration of Ic a , L recovery can result in fluctuation of cell membrane potentials during repolarization phase 2 (i.e., phase 2 EAD) that is capable of initiating TdP. Therefore, the present invention provides novel methods to screen test compounds for their effect on this intrinsic balance. The methods can be practiced according to the details below.
  • the present invention can be applied in a range of applications.
  • One application of particular value to researchers and drug developers is to test an existing or candidate pharmaceutical compound for its effect on ventricular repolarization and Ic a , L recovery.
  • the methods of the present invention can assist in the identification of compounds that are likely to give rise to a cardiac arrhythmia. This knowledge can identify and/or minimize the risk to a patient taking such pharmaceuticals that the patient will suffer a cardiac arrhythmia, ventricular defibrillation, or other LQT-related injury.
  • the methods of the present invention are therefore useful for drug design and screening.
  • the methods of the present invention can be used to screen drug candidates before they are approved for patient use or reach the marketplace.
  • the methods can be used in preclinical or clinical screening.
  • a drug designer or researcher can identify a candidate pharmaceutical that is likely to give rise to a proarrhythmic risk and, if desired, subject the compound to appropriate additional testing, or optionally remove the candidate from the research program. This can save a drug developer time and money by identifying those candidate compounds that are not worthy of pursuing in clinical trials.
  • suitable warning to medical practitioners and patients can be provided, based on data derived from the methods of the present invention.
  • the methods of the present invention can also be used to screen drugs already approved for patient use and in the marketplace. In this context, the methods can be employed to identify drugs that can pose a risk of TdP and can be marked as such. The methods of the present invention offer benefit not only to those developing drugs, but those to whom these and other drugs are administered. Ultimately, the methods of the present invention offer the ability to prevent the injury or even death of a patient.
  • Test compound or “candidate compound” are used synonymously herein, and refer to any molecule that can be analyzed using the methods of the present invention.
  • Candidate compounds to be tested by the methods of the present invention include purified molecules, substantially purified molecules, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material.
  • Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof.
  • Biomolecules include, without limitation, proteins, poplypeptides, nucleic acids, lipids, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof.
  • Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized de novo.
  • Test compounds can be defined in terms of structure or composition, or can be undefined.
  • the compound can be an isolated product of unknown structure, a mixture of several known products, or an undefined composition comprising one or more compounds.
  • undefined compositions include cell and tissue extracts, growth medium in which prokaryotic, eukaryotic, and archaebacterial cells have been cultured, fermentation broths, protein expression libraries, and the like.
  • Natural compounds can be obtained through libraries of bacterial, fungal, plant, and animal extracts, which are commercially available from a number of sources. Alternatively, the skilled artisan can generate a library of natural compounds according to methods known in the art, for example, by standard extraction and fractionation. The present invention contemplates that any library or natural compound can be modified through standard physical, chemical, biochemical, or molecular biology techniques.
  • Multiple sources of synthetic candidate compounds are also readily available to those skilled in the art. Numerous methods are available for generating random or directed synthesis, including semi-synthesis, of any number of organic or inorganic chemical compounds.
  • libraries of synthetic compounds are commercially available, for example, through Brandon Associates (Merrimakc, NH) and Aldrich Chemical Co (Milwaukee, WI).
  • the skilled artisan can generate a library of synthetic organic or inorganic chemical compounds according to methods known in the art.
  • the present invention contemplates that any library or synthetic compound can be modified through standard physical, chemical, biochemical, or molecular biology techniques.
  • cardiac tissue can be derived from any animal.
  • a functional heart is freshly isolated from an animal.
  • the animal from which the heart is isolated can be a mammal such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, and the like.
  • the heart can be isolated by any methodology acceptable in the art.
  • the whole heart can be used, or certain tissues, or cells, or heart subsections can be isolated from the heart.
  • the isolated cardiac tissue can be isolated from the left or right ventricle of the heart (“cardiac ventricular tissue"), from the left or right atrium of the heart (“cardiac atrial tissue”), or from any other subsection of the heart such as Purkinje fibers.
  • the isolated cardiac tissue can be further reduced to a cell suspension, according to any means suitable in the art.
  • Stable cells or stable cell lines in which the entire L-type Ca 2+ channel or any subunit or combination thereof of the L-type Ca 2+ channel is expressed, can also be used in the methods of the invention.
  • Stable cells or stable cell lines can be excitable or non-excitable. Such cells or cell lines can be generated de novo, according to any means suitable in the art, or can be those already established in the art. Examples of established stable cells and stable cell lines compatible with the present invention include, but are not limited to human embryonic kidney cells (HEK-293) (Peng, S et al, J. Pharmacol. Exp. Ther. 2002; 302:424-432, Traebert M et al, Eur. J. Pharmacol.
  • One aspect of the invention features methods to screen compounds to determine the potential of the compounds to induce a cardiac arrhythmia or the potential of the compounds to inhibit a cardiac arrhythmia in a subject, comprising determining the ratio of the time constant ( ⁇ ) of Ic a , L recovery in tissue expressing the L-type calcium channel or any subunit or combination thereof of the L-type calcium channel treated with a test compound to the ventricular repolarization time of cardiac tissue treated with a test compound.
  • the subject can be any animal such a mammal.
  • the subject is a human.
  • the cardiac arrhythmia can be TdP.
  • the ratio of the time constant ( ⁇ ) of Ic a , L recovery in tissue expressing the L-type calcium channel or any subunit or combination thereof of the L-type calcium channel treated with a test compound to the ventricular repolarization time of cardiac tissue treated with a test compound is determined by measuring the ventricular repolarization time of cardiac tissue treated with a test compound, measuring the recovery of the L-type calcium channel current (Ica,L) in tissue expressing the L-type calcium channel treated with the test compound, calculating the time constant ( ⁇ ) of Ic a,L recovery in tissue expressing the L-type calcium channel, and calculating the ratio of the recovery time constant ( ⁇ ) of Ic a,L to ventricular repolarization time.
  • ⁇ •
  • the effect of the test compound on ventricular repolarization and on the recovery of the L-type calcium channel current in tissue expressing the L-type calcium channel or any subunit or combination thereof of the L-type calcium channel is measured in terms of dose-dependence.
  • the cardiac tissue can be treated with a single dose of the test compound, or with multiple doses of the test compound.
  • the test compound when determining the time constant ( ⁇ ) of Ic a ,L recovery in tissue expressing the L-type calcium channel or any subunit or combination thereof of the L-type calcium channel treated with a test compound, such tissue can be treated with a single dose of the test compound, or with multiple doses of the test compound.
  • the test compound is evaluated at multiple dosages ranging from the compound's free maximal therapeutic plasma concentration (C max ) to a concentration equal to or greater than 1000-fold over the compounds' C max .
  • the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 500-fold over the compound's C raax .
  • the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 250-fold over the compound's C raax . In a still more preferred embodiment, the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 100-fold over the compound's C max . In a still more preferred embodiment, the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 50-fold over the compound's C max . In a still more preferred embodiment, the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 30-fold over the compound's C max .
  • the test compound is evaluated at multiple dosages ranging from the compound's C max to a concentration equal to or greater than 10-fold over the compound's C max .
  • C max can be determined according to any means available in the art. The skilled artisan will appreciate that such means are known and routine in the art.
  • the compound can be tested at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more concentrations.
  • the ventricular repolarization is measured at each concentration, and the effect of the test compound is compared against any control suitable in the art, such as Tyrode's solution or other balanced salt solutions without the test compound.
  • Whole blood can also serve as a control.
  • Ventricular repolarization can be measured on any cardiac tissue.
  • Non-limiting examples of such tissue include freshly isolated ventricular myocytes, freshly isolated Purkinje fibers, freshly isolated ventricular tissue such as the ventricular myocardial layer or papillary muscle, freshly isolated atrial tissue, freshly isolated ventricular or atrial wedge preparations, and the like, as would be appreciated by the skilled artisan.
  • the cardiac tissue can be derived from or isolated by any means acceptable in the art, and from any animal, as exemplified herein.
  • the ventricular action potential can be measured from the endocardium, including papillary muscle, subendocardium (M cells), or Purkinje fibers.
  • Action potential measurement can be carried out using the floating glass microelectrode recording technique in an isolated arterially-perfused ventricular wedge preparation (Yan and Antzelevitch, Circulation 1998; 98: 1921-1927), or standard glass microelectrode recording technique in isolated cardiac tissues (Antzelevitch et al., J. Am. Coll. Cardiol. 1996; 28:1836-1848).
  • monophasic action potential MAP
  • MAP can be recorded using MAP recording technique in isolated cardiac tissue or in vivo heart (Franz, Cardiovasc. Res. 1999; 41:25-40).
  • Ventricular repolarization time can also be measured by a body surface ECG, or an electrogram using bipolar or unipolar electrodes.
  • the temperature of the cardiac tissue preparation is kept in a narrow range close to the physiological temperature of the animal from which the tissue was isolated. Such a range should be within about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 degrees above or below the physiological temperature. In a more preferred embodiment, the temperature should not exceed 2 °C higher or lower than the physiological temperature of the animal. ⁇ .
  • the effect of the test compound on the Ic a,L recovery in cardiac tissue is measured under the same experimental conditions, i.e., same cardiac tissue, animal species, compound doses and dose range, temperature, and the like, as used to measure ventricular repolarization.
  • Ic a , L recovery kinetics is measured before treatment of the cardiac tissue with the test compound, and after treatment of the tissue with the test compound at the same doses used to measure ventricular repolarization.
  • Ic a , L recovery can be measured by any means suitable in the art.
  • Ic a , L recovery is measured by action potential voltage clamping techniques.
  • a preferred example of an action potential voltage clamping technique is the whole-cell voltage-clamp technique.- This technique has been described by Doerr et al. (Doerr et al., Pflugers Arch 1990; 416: 230-237).
  • the time course of Ic a , L recovery is examined using a double-pulse protocol. Since the recovery of Ic a , L from inactivation is not only time dependent but also voltage dependent, action potential configuration during phase 1 and 2 may influence Ic a,L recovery. Therefore, the assessment of the kinetics of Ic a , L recovery preferably is performed using the action potential tracing as the first voltage clamp pulse (P 1 ) followed by square pulses (P 2 ).
  • P 1 is initially held at -80 mV and steps to -40 mV to deactivate inward sodium current as well as T-type calcium current.
  • Ic a L current recovery is then recorded from holding potential of -40 mV.
  • the interpulse intervals (P 1 - P 2 ) for the voltage clamp range from 5 ms to 600 ms or longer so that Ic a , L reaches its steady state.
  • the second pulse is stepped to a potential identical to the overshoot potential of the first clamp pulse.
  • the first voltage clamp pulse can be a square pulse.
  • the kinetics of Ic a , L recovery are measured in any tissue or cell line expressing the L-type calcium channel or any subunit or combination of subunits of the channel.
  • tissue or cell line is freshly isolated, and more preferably is isolated from the same animal species in which ventricular repolarization was measured.
  • tissues that can be used in this embodiment include single ventricular myocytes and single atrial myocytes or stable cells or stable cell lines expressing the entire L- type Ca 2+ channel or any subunit or combination of subunits of the L-type calcium channel.
  • Ic a L current recovery kinetics can be measured by voltage clamping as described above.
  • the recovery time constant is calculated for each tested compound, including controls, at each dose tested.
  • the peak current ratios of Ip 2 to Ip 1 are plotted on the ordinate as a function of the interpulse interval.
  • the time constant can be calculated using computer software, such as Microcal Software, Inc., Northampton, MA.
  • the ratio of the recovery time constant ( ⁇ ) of Ic a,L to ventricular repolarization time is calculated for each test compound, including controls, and at each dose tested.
  • the ratio can be expressed as ⁇ of Ic a , L divided by the measured ventricular repolarization time that can be action potential duration or the QT interval or the duration of the electrogram.
  • the methods of the invention further comprise determining an arrhythmic risk score of a test compound.
  • the present invention contemplates that compounds that have clinically well-established QT prolongation and TdP risk profiles (for example, negative risk, low risk, moderate risk and high risk) can be tested with the inventive methods to determine their ratio of the time constant ( ⁇ ) of Ic a , L recovery to ventricular repolarization time at various doses and in various animals. Using this information, these drugs can serve as standards against which test compounds can be compared in order to determine and categorize their TdP risk score in a given species at a specified dosage level.
  • Non-limiting examples of drugs that can serve as standards for a negative risk of inducing TdP include loratadine, ciprofloxacin, clomipramine, verapamil, and fluxetine.
  • Non-limiting examples of drugs that can serve as standards for a low risk of inducing TdP include amiodarone, citalopram, flecainide, and moxifloxacin.
  • Non-limiting examples of drugs that cdn serve as standards for a moderate risk of inducing TdP include bepridil, haloperidol, sparfloxacin, and terfenadine.
  • Non- limiting examples of drugs that can serve as standards for a high risk of inducing TdP include dofetilide, sotalol, quinidine, and cisapride.
  • the inventive risk score is not intended to be limited to those compounds that have clinically well-established QT prolongation and TdP risk profiles. That is, once a test compound is evaluated according to the methods of the present invention, and the ratio of the recovery time constant ( ⁇ ) of Ic a , L to ventricular repolarization time is determined for that compound, the compound can serve as a standard against which test compounds can be compared in order to determine and categorize their TdP risk score in a given species at a specified dosage level.
  • Example 5 and Table 2 below show drugs that have been tested by the methods of the present invention that can serve as non-limiting examples of standards against which test compounds can be compared in order to determine their risk of inducing TdP at a specified dosage level.
  • a TdP risk score for a specified dose of a test compound can be determined by comparing the ratio of the time constant ( ⁇ ) of Ic a ,L recovery to the ventricular repolarization time for the test compound to the ratio of the time constant ( ⁇ ) of Ic a ,L recovery to the ventricular repolarization time of a standard having an established risk.
  • a scoring system to estimate TdP Risk of a test compound based on its effect on ventricular repolarization and the ratio of Ic a,L recovery time constant to endocardial APD 90 is shown in Table 1 below.
  • Table 1 a score of equal to or less than zero indicates no risk of TdP, a score of equal to or less than four, but greater than zero indicates a low risk of TdP, a score of equal to or less than eight, but greater than four indicates a moderate risk of TdP, and a score of eight or greater indicates a high risk of TdP.
  • the criteria, in which the ratio of the recovery time constant ( ⁇ ) of Ic a , L to ventricular repolarization time is the key element, can be adjusted according to the tissue used to take the measurements.
  • the present invention provides methods for screening a test compound for its antiarrhythmic therapeutic potential and efficacy.
  • the proarrhythmic potential of a compound is reversely proportional to the ratio of the recovery time constant ( ⁇ ) of Ic a , L to ventricular repolarization time. In other words, a smaller ratio corresponds to a higher risk that the compound induces TdP.
  • the antiarrhythmic potential of a compound is directly proportional to repolarization time and the ratio of the recovery time constant ( ⁇ ) of Ic a ,L to ventricular repolarization time.
  • a greater ratio plus a longer repolarization time corresponds to a higher antiarrhythmic potential of the compound.
  • the wavelength of the reentrant impulse should be significantly less than the length of the reentrant circuit so that a fully excitable gap is present between the crest and the tail of the reentrant wavefront for the maintenance of circus movement.
  • the wavelength of the reentrant impulse is equal to the conduction velocity times the effective refractory period of myocardial muscle in the pathway that is influenced by repolarization time and recovery of inward currents (Kowey and Yan, Heart Rhythm 2005; in press).
  • the present invention is, therefore, useful not only in screening test compounds for their capacity to cause TdP, but also in screening test compounds for their efficacy in the inhibition of cardiac arrhythmias such as atrial fibrillation and monomorphic ventricular tachycardia [0057]
  • cardiac arrhythmias such as atrial fibrillation and monomorphic ventricular tachycardia
  • Dofetilide at 0.003 ⁇ M, a concentration close to its IC 50 to block the rapidly activating delayed rectifier potassium current (I ⁇ r ) 3 markedly increased rabbit endocardial APD 90 from 252 ⁇ 8 to 443 ⁇ 27 ms (76%, n 8, ⁇ 0.01), leading to the development of EAD in seven of eight rabbit left ventricular wedge preparations.
  • Rabbit Ventricular Wedge Preparation Female rabbits weighting 2.5-3 kg were anticoagulated with heparin and anesthetized with pentobarbital (30-35 mg/kg, Lv.). The chest was opened via a left thoracotomy, and the heart was excised and placed in a cardioplegic solution consisting of cold (4 0 C) normal Tyrode's solution. Transmural wedges with dimensions of approximately 1.5 cm wide and 2-3 cm long were dissected from the left ventricle. The wedge tissue was cannulated via the circumflex or left main artery and perfused with cardioplegic solution.
  • the preparation was then placed in a small tissue bath and arterially perfused with Tyrode's solution containing 4 niM K + buffered with 95% O 2 and 5% CO 2 (T: 35.7 ⁇ 0.1 0 C, perfusion pressure: 35-45 rnmHg).
  • the ventricular wedge was allowed to equilibrate in the tissue bath until electrically stable, approximately one hour.
  • the preparations were stimulated at basic cycle lengths (BCL) of 1000 and 2000 ms using bipolar silver electrodes insulated except at the tips and applied to the endocardial surface.
  • BCL basic cycle lengths
  • ECG signal was recorded in all experiments using extracellular silver/silver chloride electrodes placed in the Tyrode's solution bathing the preparation. Electrodes were placed 1.0 to 1.5 cm from the epicardial and endocardial surfaces, along the same vector as the transmembrane recordings (Epi:"+" pole).
  • the QT interval was defined as the time from the onset of the QRS to the point at which the final downslope of the T wave crosses the isoelectric line.
  • Transmembrane action potentials were recorded simultaneously from epicardial and endocardial sites of the rabbit left ventricular wedge preparations. The action potential duration was measured at 90 % repolarization (APD 90 ).
  • QT interval in the ventricular wedge preparations quinidine, sotalol, bepridil, haloperidol, moxifloxacin, flecainide, citalopram, loratadine, fluxetine and verapamil.
  • Each of these ten compounds was tested in 4 doses ranging from their free therapeutic plasma C max to the concentration closely to or greater than 30- fold over the C max .
  • After the wedge preparations were perfused with normal Tyrode's solution for one hour in the chamber, control transmembrane action potentials and a transmural ECG were recorded. The preparations were then switched to Tyrode's solution containing a test compound with each dose for 20 minutes.
  • Single rabbit ventricular myocytes were isolated using the enzymatic digestion method as described in Rials et ah, Circulation 1997; 96:1330-1336.
  • the rabbit heart was initially perfused with 500 ml Ca 2+ -free solution (in mM: NaCl 125, KCl 3.5, KH 2 PO 4 1.5, MgCl 2 1, NaHCO 3 20, glucose 10) gassed with 95% O 2 -5% CO 2 at 37 0 C at a rate of 30 ml/min with a peristaltic pump.
  • the heart was then perfused with 80 ml enzyme solution (48 mg collagenase (type II), 12 mg hyaluronidase, 80 mg BSA 5 and 300 mg taurine to the Ca 2+ -free solution).
  • the enzyme solution passed through the heart a single time before 40 ml, and recirculation after 40 ml.
  • 7 mg protease XIV was added to the enzyme solution, and the heart was perfused for an additional 10 minutes.
  • a thin layer ( ⁇ 1.5 mm) of tissue was dissected from endocardial surface of the middle 2/3 of left ventricular free wall (excluding apex and base).
  • recovery medium in mM: potassium glutamate 80, K 2 HPO 4 20, KCl 20, MgCl 2 5, K 2 EGTA 0.5, Na 2 ATP 2, Na-pyravate 5, creatine 5, taurine 20, glycine 10, glucose 10, and HEPES 5, DNase I 0.05 mg/ml. Twenty minutes after dispersion, cells was transferred to Tyrode's solution (in niM
  • Ica,L was recorded in rabbit single ventricular myocytes at 35.7 ⁇ 0.1°C using the whole-cell voltage-clamp technique as described by Linz and Meyer, Pflugers Arch. 2000; 439:588-599.
  • the time course of ICa 1 L recovery was examined using a double-pulse protocol. Since the recovery of Ic a ,L from inactivation is not only time dependent but also voltage dependent, action potential configuration during phase 1 and 2 may influence Ic a,L recovery.
  • the action potentials were first recorded from the endocardium of the rabbit left ventricular wedge preparation at a basic cycle length of 2000 ms before and after exposure to a test compound.
  • the action potential tracing (P 1 ) and subsequent squire pulses (P 2 ) were then used for voltage clamp from holding potential of -40 mV.
  • the interpulse intervals for the voltage clamp ranged from 5 ms to 600 ms.
  • the peak current ratios of Ip 2 to Ip 1 were plotted on the ordinate as a function of the interpulse interval.
  • the ratio of Ic a , L recovery time constant to endocardial APD 90 under control and after exposure to one of the test compounds described above was determined as follows: the ratio equals ⁇ (ms)/ APD 90 (ms).
  • the relative TdP risk score was then calculated based on the change in the QT interval and the ratio according to the criteria listed in Table 1.
  • Antiarrhythmic potential of a test compound is proportional not only to its effect to prolong cardiac repolarization but also on the ratio of Ic a , L recovery time constant to repolarization.
  • the L-type calcium channel current (Ica,L) was measured both before and after exposure to quinidine, sotalol, bepridil, haloperidol, moxifloxacin, flecainide and citalopram. and compared to exposure with control compounds such as loratadine, fluxetine and verapamil.
  • An exemplary example of these measurements, carried out for sotalol, is shown in Figure 5.
  • sotalol a QT prolonging drug with a high TdP risk in humans, reduced the ratio of Ic a,L recovery time constant to endocardial APDg 0 from the control of 0.448 to 0.325 at dose of 30 ⁇ M, a concentration only 2.9-fold higher this drug's free therapeutic plasma C max for humans.
  • TdP scores of test compounds were calculated, and the calculated scores at each dose are shown in Figure 7.
  • the relative TdP are compared among quinidine, sotalol, bepridil, haloperidol, moxifloxacin, flecainide, citalopram, loratadine, fluxetine and verapamil.
  • the maximal TdP score in the testing concentration range is ranked from high to low: quinidine (11.75 ⁇ 0.22), sotalol (lO.OO ⁇ O.OO), bepridil (7.50 ⁇ 0.26), haloperidol (4.20 ⁇ 0.58), moxifloxacin (4.00 ⁇ 0.00), flecainide (1.25 ⁇ 0.22), citalopram (1.25 ⁇ 0.22), fluxetine (O.OO ⁇ O.OO), loratadine (-0.25 ⁇ 0.22) and verapamil (-2.50 ⁇ 0.26).
  • quinidine 11.75 ⁇ 0.22
  • sotalol sotalol
  • bepridil 7.50 ⁇ 0.26
  • haloperidol (4.20 ⁇ 0.58)
  • moxifloxacin (4.00 ⁇ 0.00
  • flecainide (1.25 ⁇ 0.22)
  • citalopram (1.25 ⁇ 0.22)
  • fluxetine O.OO ⁇ O.OO
  • loratadine -0.25 ⁇ 0.22
  • verapamil -2.50 ⁇

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Abstract

L'invention concerne des méthodes de criblage de composés pour leur potentiel à induire ou à inhiber une arythmie cardiaque. Les méthodes de l'invention consistent à déterminer le rapport entre la constante de temps (τ) de récupération Ica,L dans l'expression tissulaire du canal calcique de type L ou de toute sous-unité ou combinaison associée du canal calcique de type L traité avec un composé d'essai et le temps de repolarisation ventriculaire des tissus cardiaques traités avec un composé d'essai. Les méthodes de l'invention consistent également à déterminer un score de risque arythmique pour une dose spécifiée d'un composé d'essai.
PCT/US2006/029832 2005-08-02 2006-07-31 Methodes de criblage de composes pour le risque proarhythmique et l'efficacite antiarhythmique WO2007016529A2 (fr)

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